WO2022153157A1 - A crystalline form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 h-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1,3-diol free base - Google Patents

Abstract

The present invention relates to a substantially pure crystalline 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1,3-diol, to pharmaceutical compositions comprising said substantially pure crystalline form, to methods of 5 preparing and using said substantially pure crystalline form, and to compositions for the treatment of abnormal cell growth, including cancer, in a subject.

Description

A CRYSTALLINE FORM OF 2-((4-AMINO-2-(ETHOXYMETHYL)-6,7-DIMETHYL-1 H- IMIDAZO[4,5-C]PYRIDIN-1-YL)METHYL)-2-METHYLPROPANE-1 ,3-DIOL FREE BASE

FIELD OF THE INVENTION

This invention relates to a substantially pure crystalline form of 2-((4-amino-2- (ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base, to methods useful for the production and isolation of such crystalline form, to pharmaceutical compositions including them, and to methods of using such crystalline form and compositions in the treatment of abnormal cell growth in mammals, especially humans.

BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize structurally conserved molecules that are derived from and unique to pathogens, referred to as pathogen-associated molecular patterns (PAMPS). As such, TLRs function in the mammalian immune system as front-line sensors of pathogen-associated molecular patterns, detecting the presence of invading pathogens (Takeuchi and Akira 2010 Cell 140:805-820). TLR engagement in sentinel immune cells causes biosynthesis of selected cytokines (e.g., type I interferons), induction of costimulatory molecules, and increased antigen presentation capacity. These are important molecular mechanisms that activate innate and adaptive immune responses. Accordingly, agonists and antagonists of TLRs find use in modulating immune responses. TLR agonists are typically employed to stimulate immune responses, whereas TLR antagonists are typically employed to inhibit immune responses (Gosu et al 2012. Molecules 17:13503-13529).

The human genome contains 10 known TLRs, of these TLR3, TLR7, TLR8, and TLR9 recognize nucleic acids and their degradation products. The distribution of TLR7, TLR8, and TLR9 is restricted to the endosomal compartments of cells and they are preferentially expressed in cells of the immune system. In the activated dimeric receptor configuration TLR7 and TLR8 recognize single strand RNA at one ligand binding site and the ribonucleoside degradation products guanosine and uridine, respectively, (as well as small molecule ligands with related structural motifs) at a second ligand binding site (Zhang et al 2016 Immunity 45(4);737-748: Tanji et al 2015 Nat Struct Mol Biol 22: 109-1 15).

Some small-molecule TLR7 or TLR8 agonists have been identified. Those agonists can be grouped into purine-like molecules, such as 7-thia-8-oxoguanosine (TOG, isatoribine) or the imidazoquinoline-based compounds such as imiquimod. Imiquimod is so far the only approved TLR7 agonist, marketed as a 5% cream (Aldara). It generates approximately 80% 5-year clearance of superficial basal cell carcinomas, which is the most common cancer worldwide, thus demonstrating the importance of TLR7 agonists in cancer immunotherapy. The functional expression of TLR7 appears to be restricted to specific immune cells. Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of interferon a/p, which plays essential functions in the control of the adaptive immune response (Bao and Liu 2013 Protein Cell 4:40-5).

Engagement of TLR8 in myeloid dendritic cells, monocytes and monocyte-derived dendritic cells induces a prominent pro-inflammatory cytokine profile, characterized by increased production of tumor necrosis factor-a, interleukin-12, and IL-18 (Eigenbrod et al J Immunol, 2015, 195,1092-1099).

Small molecule TLR agonists have also been investigated for use as vaccine adjuvants (Dowling, ImmunoHorizons 2018, 2(6) 185-197).

Thus, virtually all major types of monocytic and dendritic cells can be activated by agonists of TLR7 and TLR8 to become highly effective antigen-presenting cells, thereby promoting an effective innate and adaptive immune response. Most antigen presenting cell types express only one of these two receptors, accordingly small molecules with potent agonist activity against both TLR7 and TLR8 receptors are potentially more effective immune adjuvants than TLR7 agonists alone.

Thus, a TLR7/TLR8 (TLR7/8) small molecule agonist with dual bioactivity could provide further benefit over a more selective TLR7 agonist and would cause innate immune responses in a wider range of antigen presenting cells and other key immune cell types, including plasmacytoid and myeloid dendritic cells, monocytes, and B cells (van Haren et al 2016 J Immunol 197:4413- 4424; Ganapathi et al 2015 Pios One 10(8). e0134640). Such potent dual TLR7/8 agonists may also be effective in stimulating effective anti-tumor responses in cancer (Singh et al 2014 J. Immunol 193 4722-4731 : Sabado et al 2015 Cancer Immunol Res 3 278-287, Spinetti et al 2016 Oncoimmunol 9;5(11):e1230578: Patil et al 2016 Mini Rev Med Chem 16:309-322).

Despite the success of Imiquimod (Aldera) in treating superficial basal cell carcinoma, there remains a need for not only more potent TLR7 agonists, but also balanced, potent TLR7/8 agonists to expand treatment options for patients for various cancers. These treatment options could be local administrations which would deliver the drug to the tumor directly, whilst limiting systemic side effects. Alternatively, systemically administered TLR7 agonists or TLR7/8 agonists would have the advantage of being able to reach difficult to administer tumors as well as multiple tumors, through the systemic circulation.

A potent and balanced TLR7/8 dual agonist 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl- 1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol (referred to herein as PF- 07225570) has been disclosed in PCT Publication No. WO 2021/009676 A1 , which is hereby incorporated for all purposes. PF-07225570 has the structure:

Preparation of PF-07225570 as disclosed in Example 2 of WO 2021/009676 A1 provided a white solid, which was found to be a mixture of crystalline forms that comprises predominantly a crystalline form referred to herein as Form 1 . The percentage of the crystalline Form 1 in the white solid varied between batches. Preparation of a pure and homogeneous crystalline form is critical in the quality control of drug product manufacturing process and drug pharmaceutical compositions. Therefore, there is a need to identify and reproducibly prepare a pure crystalline form for PF-07225570.

SUMMARY OF THE INVENTION

The present invention provides a substantially pure anhydrous crystalline form of 2-((4- amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane- 1 ,3-diol free base (Form 2), pharmaceutical compositions comprising Form 2, and methods of making and using Form 2 for the treatment of cancer.

In one aspect, Form 2 has a powder X-ray diffraction (PXRD) pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ± 0.2 °20.

In one aspect, Form 2 has at least 95% pure crystalline form. In one preferred aspect, Form 2 has at least 98% pure crystalline form.

In one aspect, Form 2 has a PXRD profile that is free of characterization peaks of Form 1 , i.e., free of PXRD peaks of 7.5, 9.3, and 15.7 °20 ± 0.2 °20, with relative intensity over 5%. In one preferred aspect, Form 2 has a PXRD profile that is free of characterization peaks of Form 1 ,

1.e., free of PXRD peaks of 7.5, 9.3, and 15.7 °20 ± 0.2 °20, with relative intensity over 2%.

The present invention also provides, in part, methods for preparing Form 2, and compositions of the invention, and methods of using the foregoing alone or in combination with additional anticancer therapeutic agents.

In another aspect, the invention provides a pharmaceutical composition comprising Form

2, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises two or more pharmaceutically acceptable carriers and/or excipients.

In another aspect, the invention also provides therapeutic methods and uses comprising administering Form 2.

In another aspect, the invention provides a method for the treatment of abnormal cell growth, in particular, cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Form 2. Form 2 may be administered as single agent or may be administered in combination with other anti-cancer therapeutic agents, including standard of care agents appropriate for the particular form of cancer. This also includes use of Form 2, in the manufacture of a medicament for treating abnormal cell growth, in particular, cancer, in a subject in need thereof. In another aspect, the invention provides Form 2 for use as a medicament, in particular a medicament for the treatment of abnormal cell growth, such as cancer.

In yet another aspect, the invention provides the use of Form 2 for the manufacture of a medicament for the treatment of abnormal cell growth, such as cancer, in a subject.

In one aspect, the invention provides a method of preparing the substantially pure anhydrous crystalline Form 2, wherein the method comprises treating 2-(ethoxymethyl)-N,N- bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5- c]pyridin-4-amine in a solvent system comprising a solvent with a boiling point of at least 70 °C, with an acid to provide 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1- yl)methyl)-2-methylpropane-1 ,3-diol as a solid, wherein the solid has Form 2 as predominant crystalline form. In another aspect, the method further comprises dissolving the solid having Form 2 as predominant crystalline form in a solvent system comprising a CMO alcohol to form a solution and heating the solution at an elevated temperature; and followed by adding a solvent comprising a C4-10 alkane to the solution to provide the substantially pure anhydrous crystalline Form 2.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an illustrative PXRD pattern of Form 2, as prepared in Example 3, carried out on a Bruker D8 Endeavor diffractometer equipped with a copper (Cu) radiation source.

FIG. 2 shows a single crystal of Form 2.

FIG. 3 shows an illustrative PXRD pattern of PF-07225570, as prepared in Example 2, carried out on a Bruker D8 Endeavor diffractometer equipped with a Cu radiation source.

FIG. 4 shows a single crystal of Form 1 mixed with Form 2 as impurity.

FIG. 5 shows an illustrative PXRD pattern of the amorphous form of PF-07225570 free base.

FIG. 6 provides Raman spectrum of the amorphous form of PF-07225570 free base.

DETAILED DESCRIPTION OF THE INVENTION

As provided, the invention concerns Form 2, which is a substantially pure anhydrous, crystalline form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1- yl)methyl)-2-methylpropane-1 ,3-diol free base.

The present invention may be understood more readily by reference to the following detailed description of additional embodiments of the invention. The preparations of Form 2 are not intended to be limiting. It is to be understood that the terminology used herein is provided for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.

As used herein, the singular form "a", "an", and "the" include plural references unless indicated otherwise. For example, "a" substituent includes one or more substituents. As used herein, the term “Ci-w alcohol” means a straight or branched C1-10 alcohol.

As used herein, the term “C4-10 alkane” means a straight or branched C4-10 alkane.

As used herein, the term “predominant” means that in a solid comprising more than one crystalline forms, the crystalline form in the solid with the highest weight percentage is referred as the predominant component.

As used herein, the term “essentially the same” means that variability typical for a particular method is taken into account. For example, with reference to X-ray diffraction peak positions, the term “essentially the same” means that typical variability in peak position and intensity are taken into account. One skilled in the art will appreciate that the peak positions (20) will show some variability, typically as much as ± 0.2°. Further, one skilled in the art will appreciate that relative peak intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, preferred orientation, prepared sample surface, and otherfactors known to those skilled in the art and should be taken as qualitative measures only. A variability may be between ±%.

The term “crystalline” as used herein, means having a regularly repeating arrangement of molecules or external face planes. Crystalline forms may differ with respect to thermodynamic stability, physical parameters, x-ray structure and preparation processes.

The term “crystalline purity” for a specific crystalline form as used herein means the weight percentage of the specific crystalline form in all possible crystalline forms of a solid compound. For example, if a solid compound consists of 90 grams of a crystalline form A and 10 grams of a crystalline form B, the crystalline purity of the crystalline A is 90%.

The term “substantially pure” as used herein for the crystalline purity of a crystalline compound means the crystalline compound has at least 95% crystalline purity. Preferably, the substantially pure crystalline compound has at least 98% crystalline purity. Alternatively, “substantially pure” as used herein for the crystalline purity of a crystalline compound means that there is no identified characterization peak of other crystalline forms with a relative intensity over such as 5% in PXRD under the same condition to generate PXRD as disclosed in this invention. In a preferred aspect, there is no identified characterization peak of other crystalline forms with a relative intensity over such as 2% in PXRD under the same condition to generate PXRD as disclosed in this invention.

As used herein, "subject" refers to a human or animal subject. When the subject is a human, the subject may also be referred to as a “patient”.

The term “treat” or “treating” a cancer as used herein means to administer a compound of the present invention to a subject having cancer, or diagnosed with cancer, to achieve at least one positive therapeutic effect, such as, for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastases or tumor growth, reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject.

The terms “treatment regimen”, “dosing protocol” and “dosing regimen” are used interchangeably to refer to the dose and timing of administration of each compound of the invention, alone or in combination with another therapeutic agent.

“Abnormal cell growth”, as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). Abnormal cell growth may be benign (not cancerous), or malignant (cancerous).

The terms “cancer” or “cancerous” refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. Cancer includes primary cancerthat originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a patient with a history of previous cancer of a different type from the second primary cancer. Cancer includes solid tumors named for the type of cells that form them, cancer of blood, bone marrow, or the lymphatic system. Examples of solid tumors include sarcomas and carcinomas. Cancers of the blood include leukemia, lymphoma and myeloma. Additional examples of cancer include blastomas and an actinic keratosis. Cancer also includes primary cancer or metastases of a site selected from the group consisting of oral cavity, digestive system, respiratory system, skin, breast, genital system, urinary system, ocular system.

Example 2 disclosed in WO 2021/009676 A1 was made and was referred as a white solid. It has been later found that the white solid prepared is not a pure crystalline form although chemically pure. For a pharmaceutical material, a pure and homogeneous crystalline form is critical in the quality control of drug product manufacturing process and drug product compositions. Although extensive effort to prepare pure Form 1 has not been successful, surprisingly, with a unique process, including a unique combination of solvent system, temperature control (See Step 6 for preparing Form 2), the present disclosure provides a substantially pure crystalline form referred as Form 2, which has a powder X-ray diffraction (PXRD) pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ± 0.2 °20.

It has been found that at least two factors have contributed to the substantially pure crystalline form of Form 2. The first factor is the different solvent in the step of converting 2- (ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5-trimethyl-1 ,3-dioxan-5- yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine to 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H- imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol. When dichloromethane is used, Form 1 is the predominant final solid form and it always has another solid form as impurity which cannot be removed. When the solvent is toluene, surprisingly, the predominant solid form is the form of Form 2. With further treatment of the material in 1 -butanol and followed by n-heptane, the material was enriched to pure or substantially pure crystalline form of Form 2. In one embodiment, the invention provides Form 2, which is a substantially pure crystalline form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1- yl)methyl)-2-methylpropane-1 ,3-diol free base, having the structure:

Form 2 is an anhydrous, crystalline solid having a powder X-ray diffraction (PXRD) pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ± 0.2 °20.

Form 2 as prepared in this disclosure has at least 95% pure crystalline form. In one preferred aspect, Form 2 has at least 98% pure crystalline form.

In one aspect, Form 2 of the present invention has a PXRD profile that is free of characterization peaks of Form 1 , i.e., free of PXRD peaks of 7.5, 9.3, and 15.7 °20 ± 0.2 °20. Another embodiment of Form 2 can be further described as an anhydrous crystalline having at least 99% chemical purity. Another embodiment of Form 2 can be further described as an anhydrous crystalline form having at least 99.6% chemical purity.

Another embodiment of the invention provides a pharmaceutical composition comprising Form 2 and a pharmaceutically acceptable carrier or excipient.

Another embodiment of the invention provides a method for the treatment of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Form 2.

Cancers to be treated include squamous cell carcinoma, basal cell carcinomas, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, uterine cancer, bladder cancer, including non-muscular invasive bladder cancer, hepatoma, breast cancer, and head and neck cancer.

More particular examples of cancers to be treated include basal cell carcinomas, smallcell lung cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, ovarian cancer, colorectal cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, pancreatic cancer, bladder cancer (non-muscular invasive bladder cancer), hepatoma, breast cancer, and head and neck cancer.

Another embodiment of the invention concerns Form 2 for treatment of non-muscular invasive bladder cancer. Another embodiment of the invention concerns Form 2 for use in the treatment of cancer in a subject in need thereof.

Another embodiment of the invention concerns Form 2 for use in the treatment of cancer, wherein said treatment comprises the administration of an additional therapeutic agent.

In one aspect, the invention provides a method of preparing the substantially pure anhydrous crystalline Form 2, wherein the method comprises treating 2-(ethoxymethyl)-N,N- bis(4-methoxybenzyl)-6,7-dimethyl-1-((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5- c]pyridin-4-amine in a solvent system comprising a solvent with a boiling point of at least 70 °C, with an acid to provide 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1- yl)methyl)-2-methylpropane-1 ,3-diol as a solid, wherein the solid has Form 2 as predominant crystalline form.

In another aspect, the method further comprises dissolving the solid having Form 2 as predominant crystalline form in a solvent system comprising a Ci-w alcohol to form a solution and heating the solution at an elevated temperature; and followed by adding a solvent comprising a C4-10 alkane to the solution to provide the substantially pure anhydrous crystalline Form 2.

In one preferred aspect, the invention provides a method of preparing the substantially pure anhydrous crystalline Form 2, wherein the method comprises a) treating 2-(ethoxymethyl)-

N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H- imidazo[4,5-c]pyridin-4-amine in a solvent system comprising toluene with an acid to provide 2- ((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2- methylpropane-1 ,3-diol as a solid, wherein the solid has Form 2 as predominant crystalline form; and b) dissolving the solid in a solvent comprising 1 -butanol to form a solution and heating the solution at an elevated temperature; and followed by adding n-heptane to the solution to provide the substantially pure anhydrous crystalline Form 2.

In one aspect, Form 2 is prepared by the foregoing methods.

Embodiments (EB)

EB1 . A substantially pure anhydrous crystalline form of 2-((4-amino-2-(ethoxymethyl)-6,7- dimethyl-1/7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base, having a powder X-ray diffraction (PXRD) pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ±

O.2 °20.

EB2. The substantially pure anhydrous crystalline form of EB1 , having a PXRD pattern further comprising a peak value of 21 .6 °2Q ± 0.2 °2Q.

EB3. The substantially pure anhydrous crystalline form of EB1 or EB2, having at least 95% crystalline purity.

EB4. The substantially pure anhydrous crystalline form of EB3, having at least 98% crystalline purity. EB5. The substantially pure anhydrous crystalline form of any one of EB1 to EB4, wherein the PXRD pattern is free of peaks with a relative intensity over 5% selected from the group consisting of 7.5, 9.3, and 15.7 °20 ± 0.2 °20.

EB6. A pharmaceutical composition comprising the substantially pure anhydrous crystalline form of any one of EB1 to EB5, and a pharmaceutically acceptable carrier or excipient.

EB7. A method for the treatment of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the substantially pure anhydrous crystalline form of any one of EB1 to EB5.

EB8. The method of EB7, wherein the substantially pure anhydrous crystalline form is administered with at least one additional therapeutic agent.

EB9. The substantially pure anhydrous crystalline form of any one of EB1 to EB5, for use in the treatment of cancer in a subject in need thereof.

EB10. A method of preparing the substantially pure anhydrous crystalline form of any one of EB1 to EB5, wherein the method comprises treating 2-(ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7- dimethyl-1-((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine in a solvent system comprising a solvent with a boiling point of at least 70 °C, with an acid to provide 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2- methylpropane-1 ,3-diol as a solid, wherein the anhydrous crystalline form of any one of EB1 to EB5 is predominant crystalline form in the solid.

EB1 1. The method of EB10, wherein the method further comprises dissolving the solid in a solvent system comprising a C1-10 alcohol to form a solution and heating the solution at an elevated temperature; and followed by adding a solvent comprising a C4-10 alkane to the solution to provide the substantially pure anhydrous crystalline of any one of EB1 to EB5.

EB12. The method of EB1 1 , wherein said CMO alcohol is 1 -butanol.

EB13. The method of EB1 1 or EB12, wherein said C4-10 alkane is n-heptane.

EB14. The method of any one of EB10 to EB13, wherein said solvent with a boiling point of at least 70 °C is toluene.

EB15. The substantially pure anhydrous crystalline form of any one of EB1 to EB5, wherein the substantially pure anhydrous crystalline form is prepared by the method of any of EB10 to EB14.

Chemical Synthesis

The following synthetic description provides details regarding the preparation of Form 2.

The skilled person will appreciate that the experimental conditions set are illustrative of suitable conditions for effecting the transformations shown.

In addition, the skilled person will appreciate that it may be necessary or desirable at any stage in the synthesis of Form 2 to protect one or more sensitive groups, to prevent undesirable side reactions. Conventional processes are known to the skilled person. See, for example, those described in 'Greene’s Protective Groups in Organic Synthesis' by Theodora W Greene and Peter G M Wuts, third edition, (John Wiley and Sons, 1999), in particular chapters 7 (“Protection forthe Amino Group”) and 2 (“Protection for the Hydroxyl Group, Including 1 ,2- and 1 ,3-Diols”), incorporated herein by reference, which also describes methods for the removal of such groups.

EXAMPLES

Except where otherwise noted, reactions were run under an atmosphere of nitrogen. Chromatography on silica gel was carried out using 250-400 mesh silica gel using pressurized nitrogen (~10-15 psi) to drive solvent through the column (“flash chromatography”). Where indicated, solutions and reaction mixtures were concentrated by rotary evaporation under vacuum.

¹H Nuclear magnetic resonance (NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical shifts (6) are given in parts-per-million downfield from tetramethylsilane (for ¹H-NMR) using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The following abbreviations have been used for common solvents: CDCI₃, deuterochloroform; d₆-DMSO, deuterodimethylsulfoxide; and CD3OD, deuteromethanol. Where appropriate, tautomers may be recorded within the NMR data; and some exchangeable protons may not be visible.

Mass spectra, MS (m/z), were recorded using either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI).

The nomenclature is written as described by IUPAC (International Union of Pure and Applied Chemistry generated within Perkin Elmers Chemdraw 18.0.

In the preparations that are set out herein, the following the abbreviations apply: aq is aqueous;

°C is degrees Celsius;

CO2 is carbon dioxide;

DMAC is dimethylacetamide;

DMSO is dimethyl sulphoxide;

EtOAc is ethyl acetate; g is gram;

HCI is hydrochloric acid;

HPLC is high pressure liquid chromatography;

H₂O is water;

Hr or hr is hour;

L is litre;

LCMS is liquid chromatography mass spectrometry;

M is molar; mg is milligram; MHz is mega Hertz; min is minutes; mL or ml is milli litre; mmol is millimole; mol is mole;

MS m/z is mass spectrum peak;

NH(PMB)2 is bis(4-methoxybenzyl)amine;

NMR is nuclear magnetic resonance;

Pd/C is palladium on carbon; pH is power of hydrogen; ppm is parts per million; psi is pounds per square inch;

Rt is retention time; pL is microlitre; pmol is micromol

Wherein preparative TLC or silica gel chromatography have been used, one skilled in the art may choose any combination of solvents to purify the desired compound.

Example 1 : Synthesis 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H-imidazo[4,5- c]pyridin-1 -yl)methyl)-2-methylpropane-1 ,3-diol (PF-07225570)

Step 1 : Synthesis of 5,6-dimethyl-3-nitropyridine-2,4-diol

To 5,6-dimethylpyridine-2,4-diol (40.0 g, 287 mmol) (Org. Lett., 2003, 5 (25), pp 4779- 4782) at 18°C was added concentrated sulfuric acid (174 ml). The reaction was cooled to 0°C at which point nitric acid (68-70%, 45.8 ml) was added over 1 ,5hrs, maintaining internal temperature below 10°C. After addition was complete the reaction was stirred at 10°C for 30min. This reaction was combined with a second reaction from 40 g 5,6-dimethylpyridine-2,4-diol. The combined reaction mixture was poured into ice-water (2 L). The yellow precipitate was collected by filtration and washed with water (5 x 200 ml) and MTBE (5 x 100 ml). The collected solids were dried under vacuum to provide the title compound as a yellow solid. Combined Yield: 50 g, 271 .7 mmol, 47% yield based on 80 g starting pyridine. ¹H NMR (400 MHz, DMSO-cfe) 12.34 (br s, 1 H), 11.90 (br s, 1 H), 2.21 (s, 3H), 1.90 (s, 3H).

Step 2: Synthesis of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-3-nitro-N4-((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)pyridine-2,4-diamine

To a solution of 5,6-dimethyl-3-nitropyridine-2,4-diol (70.0 g, 380.1 mmol) in dichloroethane (1.4 L) cooled to 0°C was added triethylamine (80.8 g, 798 mmol). Trifluoromethanesulfonic anhydride (220 g, 779 mmol) was added over 30 min at 0°C. The reaction was stirred at 0°C for 1.5 hr. Triethylamine (42.3 g, 418 mmol) was added followed by portionwise addition of (2,2,5-trimethyl-1 ,3-dioxan-5-yl)methanamine (72.6 g, 456 mmol) (Prepared from Organic & Biomolecular Chemistry, 14(2), 483-494; 2016). The reaction was stirred at 0 °C for 20min then stirred at 15 °C for 18hrs. The reaction was cooled to 0 °C at which point triethylamine (115 g, 1.14 mol) was added followed by Bis(4-methoxybenzyl)amine (127 g, 494 mmol). The reaction was then stirred at 50°C for 12 hrs. The solvent was removed and the residue was purified via silica gel column chromatography (Petroleum EtherEtOAc gradient 0- 10%). Product fractions were collected and evaporated to 10% volume. Solids were collected via filtration and filter cake washed with petroleum ether (3 x 50 ml). The filtrate was concentrated and purified via silica gel column chromatography (Petroleum EtherEtOAc gradient 0-10%). Product fractions were collected and evaporated to 10% volume. Solids were collected via filtration and the filter cake washed with petroleum ether (3 x 20 ml), providing the title compound as a yellow solid. Yield: 98 g, 173.6 mmol, 45.7%. LCMS m/z 564.9 [M+H]⁺. ¹H NMR (400 MHz, CDCI₃) 6 7.05 (d, J=8.78 Hz, 4H), 6.80 (d, J=8.78 Hz, 4H), 6.47 (t, J=6.15 Hz, 1 H), 4.34 (s, 4H), 3.79 (s, 6H), 3.54-3.67 (m, 4H), 3.42 (d, J=6.02 Hz, 2H), 2.35 (s, 3H), 2.21 (s, 3H), 1.43 (s, 3H), 1.41 (s, 3H), 0.83 (s, 3H).

Step 3: Synthesis of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-N4-((2,2,5-trimethyl-1 ,3-dioxan- 5-yl)methyl)pyridine-2,3,4-triamine

To a solution of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-3-nitro-N4-((2,2,5-trimethyl- 1 ,3-dioxan-5-yl)methyl)pyridine-2,4-diamine (57.0 g, 100.9 mmol) in methanol (673 ml) was added ammonium formate (63.7 g, 1 .01 mol) and then Zinc Dust (66.0 g, 1 .01 mol). The reaction was stirred for 10 min at 15°C. The reaction mixture was filtered through Celite® and the filtrate concentrated. The residue was dissolved in EtOAc and water slowly added to form a white precipitate. The aqueous layer was extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to provide the title compound as a brown oil. Used without further purification Yield: 50.0 g, 96.4 mmol, 95.5%. LCMS m/z 535.0 [M+H]⁺.

Step 4: Synthesis of N-(2-(bis(4-methoxybenzyl)amino)-5,6-dimethyl-4-(((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)amino)pyridin-3-yl)-2-ethoxyacetamide

To a solution of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-N4-((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)pyridine-2,3,4-triamine (100 g, 192.8 mmol) in dichloromethane (1 L) was added triethylamine (97.5 g, 964 mmol, 134 ml). The reaction was cooled to 0°C at which point 2-ethoxyacetyl chloride (37.8 g, 308 mmol) was added dropwise. The ice bath was removed and the reaction stirred at 25°C for 16 hrs. The solvent was removed, and the product used without further purification. Yield: 150 g, 192.8 mmol, assumed quantitative.

Step 5: Synthesis of 2-(ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5- trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine

To a solution of crude N-(2-(bis(4-methoxybenzyl)amino)-5,6-dimethyl-4-(((2,2,5- trimethyl-1 ,3-dioxan-5-yl)methyl)amino)pyridin-3-yl)-2-ethoxyacetamide from the above reaction in ethanol (3.45 L cooled to 0°C) was added sodium hydroxide (64.4 ml, 15N aqueous). After addition the reaction was heated to reflux for 16 hrs. The reaction was cooled to 15°C at which point a white precipitate formed. The solids were filtered and the filter cake washed with water and MTBE. The white solids were dissolved in EtOAc (1 L) and the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (EtOAc: Petroleum Ether gradient 0-45%.) to provide product. This material was combined with an additional 7.2g of product from a different batch and stirred for 20min in a 2:1 solution of petroleum etherMTBE. The solids were filtered and dried on vacuum to provide the title compound as a white solid. Combined yield: 94.52 g, 157 mmol, 76% yield. LCMS m/z 603.0 [M+H]⁺. ¹H NMR (600 MHz, DMSO-c/₆) 6 7.16 (d, J=8.22 Hz, 4H), 6.83 (d, J=8.80 Hz, 4H), 4.82-5.27 (m, 6H), 4.36-4.80 (m, 4H), 3.70 (s, 6H), 3.51-3.68 (m, 2H), 3.40-3.46 (m, 2H), 2.41 (s, 3H), 2.34 (s, 3H), 1 .39 (s, 3H), 1 .34 (s, 3H), 1 .07 (t, J=7.04 Hz, 3H), 0.56 (s, 3H).

Step 6: Synthesis of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H-imidazo[4,5-c]pyridin-1- yl)methyl)-2-methylpropane-1 ,3-diol (PF-07225570)

To a solution of 2-(ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5- trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine (477 mg, 0.791 mmol) and dichloromethane (3 ml) in a vial was added concentrated HCI (7.91 mmol, 0.66 ml) dropwise. The vial was capped and the reaction was stirred at RT for 2hrs then heated at 50°C for 1 hr. Additional concentrated HCI (4.8 mmol, 0.40 ml) was added and the reaction continued heating at 50°C for 30min. The reaction was then cooled to RT and stirred for 16hrs. Water (2 ml) was added and the aqueous was washed with dichloromethane (2 x 3 ml). The aqueous was brought to pH 9 with solid sodium carbonate. The reaction was then heated to reflux and cooled to 4°C. The solids were filtered and washed with water (5ml) and ether (5ml) and dried under vacuum to provide the title compound as a white solid. Yield. 194 mg, 0.602 mmol, 76.0%. LCMS m/z 323.3 [M+H]⁺. ¹H NMR (400 MHz, DMSO-cfe) 6 5.76 (s, 2H), 4.91-5.09 (m, 1 H), 4.69-4.90 (m, 2H), 4.27-4.59 (m, 3H), 3.39-3.56 (m, 2H), 3.29-3.37 (m, 1 H, assumed, partially obscured by H₂O), 3.14-3.29 (m, 2H), 2.95-3.13 (m, 1 H), 2.41 (s, 3H), 2.29 (s, 3H), 1.06-1 .14 (m, 3H), 0.48 (s, 3H). Step 6A: Alternative Step 6 for synthesis of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H- imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol (PF-07225570)

To a solution of 2-(ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5- trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine (2.31 g, 3.83 mmol, prepared in a manner similar to that described Steps 1 to 5 of Example 2) and dichloromethane (15 mL) was added concentrated hydrochloric acid (38.3 mmol, 3.19 mL). Reaction was stirred at 20 °C for 2.5 hours. The reaction was then brought to reflux for 2 hours at which point concentrated HCI was added (3 mL) and reflux continued for 1 hour. Concentrated HCI (2 mL) was added and the reaction continued to heat at reflux for 30 min. The reaction was cooled to 20 °C and stirred for 16hrs. The reaction was then warmed to 50 °C and concentrated HCI was added (3 x 1 mL, each 1 mL portion added 20 min apart). Heating continued for an additional 20 min after addition complete then cooled to 20 °C. The reaction was diluted with water (20 mL) and the aqueous washed with dichloromethane (3 x 20 mL). The aqueous was brought to pH 9 with sodium carbonated and the volume reduced to 25 mL with a stream of nitrogen. The reaction was cooled in an ice bath and solids collected via filtration. The solids were washed with water (30 mL) and diethylether (30 mL) and the solids were dried in vacuo at 50 °C for 16 hrs. Yield. 1.08 g, 3.35 mmol, 87.4%. LCMS m/z 323.3 [M+H]⁺. ¹H NMR (400 MHz, DMSO-c/₆) 6 5.76 (s, 2H), 4.91-5.09 (m, 1 H), 4.69-4.90 (m, 2H), 4.27-4.59 (m, 3H), 3.39-3.56 (m, 2H), 3.29-3.37 (m, 1 H, assumed, partially obscured by H₂O), 3.14-3.29 (m, 2H), 2.95-3.13 (m, 1 H), 2.41 (s, 3H), 2.29 (s, 3H), 1.06- 1.14 (m, 3H), 0.48 (s, 3H).

Example 2: Alternative synthesis of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H- imidazo[4,5-c]pyridin-1-yl)methyl)-2 -methyl propane-1 ,3-diol (PF-07225570)

Step 1 (i): Synthesis of 5,6-dimethyl-3-nitropyridine-2,4-diol

This intermediate was made similarly as previously described in Step 1 of Example 1.

Step 2(i): Synthesis of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-3-nitro-N4-((2,2,5-trimethyl- 1 ,3-dioxan-5-yl)methyl)pyridine-2,4-diamine

To a round bottom flask under nitrogen was added 5,6-dimethyl-3-nitropyridine-2,4-diol (40.2 g, 218.3 mmol) in dichloromethane (1 L). To this was added triethylamine (110.0 g, 1.09 mol, 152.0 ml) and the reaction cooled to 7 °C. Triflic anhydride (126.0 g, 448 mmol, 75.3 ml) was added dropwise over 100 min. The reaction was stirred between 0 °C and 7 °C for 10 min. To this was added Triflic anhydride (6.32 g, 22.4 mmol, 3.77 ml) over 2 min and the reaction was stirred at 0 °C for 10min. (2,2,5-trimethyl-1 ,3-dioxan-5-yl)methanamine (36.5 g, 229 mmol) dissolved in dichloromethane (35 mL) was added in 2 mL portions over 12 min. The reaction was then stirred at 0 °C for 30min. Bis(4-methoxybenzyl)amine (70.2 g, 273 mmol) was added in 1 portion and the reaction was heated at reflux for 16 hrs. The reaction was then cooled to 20 °C and washed with water (2 x 600 mL). The organics were dried over anhydrous sodium sulfate, filtered and concentrated on silica gel. The silica gel was transferred to the top of an 8 inch silica plug conditions with heptane in a 2L filter funnel. The product was then eluted with Heptane/EtOAc (80/20 mix, 3.5L). The product was concentrated to a red solid that was stirred in ethanol (300 mL) for 2hrs. The solids were filtered and washed with ethanol (200 ml) and dried in vacuo to provide title compound as a yellow solid. Yield: 53.4 g, 94.57 mmol, 43.3%. LCMS m/z 564.9 [M+H]⁺. ¹H NMR (400 MHz, CDCh) 6 7.05 (d, J=8.78 Hz, 4H), 6.80 (d, J=8.78 Hz, 4H), 6.47 (t, J=6.15 Hz, 1 H), 4.34 (s, 4H), 3.79 (s, 6H), 3.54-3.67 (m, 4H), 3.42 (d, J=6.02 Hz, 2H), 2.35 (s, 3H), 2.21 (s, 3H), 1 .43 (s, 3H), 1 .41 (s, 3H), 0.83 (s, 3H). Additional material was prepared using similar conditions.

Step 3(i): Synthesis of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-N4-((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)pyridine-2,3,4-triamine

To a solution of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-3-nitro-N4-((2,2,5-trimethyl- 1 ,3-dioxan-5-yl)methyl)pyridine-2,4-diamine (51.6 g, 91 .38 mmol) in methanol (500 ml) was added ammonium formate (28.8 g, 457.0 mmol) and then Zinc Dust (29.9 g, 457.0 mol). The reaction was stirred for 5 min at 20°C then cooled to 0 °C and stirred for 20 min. The reaction mixture was filtered through Celite® with methanol (200 mL) and the filtrate concentrated. The residue was stirred in EtOAc (500 mL) for 45 min. The solids were filtered through Celite® and the filtrate concentrated to provided title compound, that was used without further purification. Yield: 48.90 g, 91 .45 mmol, 100.0%. LCMS m/z 535.5 [M+H]⁺. ¹H NMR (CHLOROFORM-d, 600 MHz) 6 7.19 (d, 4H, J=8.5 Hz), 6.79 (d, 4H, J=8.5 Hz), 4.13 (s, 4H), 3.8-3.8 (m, 8H), 3.66 (d, 2H, J=1 1.7 Hz), 3.14 (s, 2H), 2.34 (s, 3H), 2.1 1 (s, 3H), 1.47 (s, 3H), 1 .43 (s, 3H), 0.93 (s, 3H). Additional material was prepared using similar conditions.

Step 4(i): Synthesis of N-(2-(bis(4-methoxybenzyl)amino)-5,6-dimethyl-4-(((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)amino)pyridin-3-yl)-2-ethoxyacetamide

To a solution of N2,N2-bis(4-methoxybenzyl)-5,6-dimethyl-N4-((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)pyridine-2,3,4-triamine (48.9 g, 91 .45 mmol) in dichloromethane (300 mL) was added sodium bicarbonate (23.0 g, 274 mmol) and water (250 mL). The reaction was cooled to 0°C with vigorous stirring at which point 2-ethoxyacety I chloride (12.3 g, 101 mmol, 11.0 mL) was added dropwise. The ice bath was removed and the reaction stirred at 20 °C for 45 hrs. The organics were separated and the aqueous was washed with dichloromethane (1x 200 mL). The organics were combined, dried over anhydrous sodium sulfate, filtered and concentrated to provide title compound that was used without further purification. Yield: 56.8 g, 91 .49 mmol, assumed quantitative. Additional material was prepared using similar conditions.

Step 5(i): Synthesis of 2-(ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5- trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine

To a solution of N-(2-(bis(4-methoxybenzyl)amino)-5,6-dimethyl-4-(((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)amino)pyridin-3-yl)-2-ethoxyacetamide (56.8 g, 91.49 mmol) in ethanol (500 mL) was added sodium hydroxide (24.2 ml, 50 % aqueous). After addition the reaction was heated to reflux for 16 hrs. The reaction was cooled to 20 °C and concentrated to 10 % volume. The reaction was diluted with water (500 mL). The aqueous was washed with dichloromethane (2 x 400 mL) and combined organics washed with water (1 x 200 mL). The organics were dried over anhydrous sodium sulfate and filtered. The material was then combined with another batch of compound prepared in a similar manner at a similar stage of the synthesis. The combined organics were then concentrated to 15% volume. The solid that formed was filtered and washed with ethanol (250 mL) and then water (1 L). The solids were combined with the filtrate and the liquid and solids decanted away from a red slime that formed at the bottom of the flask. To the decanted heterogenous mixture was added water (1 .3 L) and the mixture stirred for 16 hrs. The solids were filtered and washed with water (1 L) then slurried in ethanol (200 mL) for 48 hrs. The solids were filtered and washed with ethanol (200 mL) and solids dried in vacuo to provide the title compound as an off-white solid. Yield: 173.5 g, 287.84 mmol, 63.8% combined yield. LCMS m/z 603.0 [M+H]⁺. ¹H NMR (600 MHz, DMSO-c/₆) 6 7.16 (d, J=8.22 Hz, 4H), 6.83 (d, J=8.80 Hz, 4H), 4.82-5.27 (m, 6H), 4.36-4.80 (m, 4H), 3.70 (s, 6H), 3.51-3.68 (m, 2H), 3.40-3.46 (m, 2H), 2.41 (s, 3H), 2.34 (s, 3H), 1.39 (s, 3H), 1.34 (s, 3H), 1.07 (t, J=7.04 Hz, 3H), 0.56 (s, 3H). Additional material was prepared using similar conditions.

Step 6(i): Synthesis of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H-imidazo[4,5-c]pyridin-1- yl)methyl)-2-methylpropane-1 ,3-diol (PF-07225570)

To a solution of 2-(ethoxymethyl)-N,N-bis(4-methoxybenzyl)-6,7-dimethyl-1 -((2,2,5- trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5-c]pyridin-4-amine (100.8 g, 167.45 mmol) and dichloromethane (500 ml) was added concentrated HCI (1 .20 mol, 100 ml) over 30 seconds. The reaction was heated at 50°C for 30 min. Additional concentrated HCI (600 mmol, 50 ml) was added and the reaction continued heating at 50 °C for 30min. Additional concentrated HCI (600 mmol, 50 ml) was added and the reaction continued heating at 60 °C for 30min. Additional concentrated HCI (600 mmol, 50 ml) was added and the reaction continued heating at 80 °C for 90 min. Additional concentrated HCI (480 mmol, 40 ml) was added and the reaction continued heating at reflux for 30 min. The reaction was cooled to 20 °C and water (30 mL) was added. The organics were removed and the aqueous was washed with dichloromethane (1 x 250 mL). The aqueous phase was placed in a 110 °C heating block. As the reaction was heating towards reflux the mixture was brought to a pH of 10 with sodium carbonate. The reaction was refluxed for 30 min then cooled to 10 °C. Solids were filtered and washed with water (1 x 300 mL) and tetra hydrofuran (1 x 50 mL). The solids were combined with additional batches prepared under a similar manner to provide 259 g total crude solids. The combined batches were stirred with tetra hydrofuran (350 mL) for 60 min at 45 °C. The reaction was cooled to 15 °C and the solids filtered and washed with tetrahydrofuran (150 mL). The solids were then stirred in tetra hydrofuran (400 mL) for 120 min at 60 °C and then cooled to 20 °C and stirred for 16 hrs. The solids were collected and stirred with water (650 mL, HPLC grade) at reflux for 7 hrs. Water (200 mL, HPLC grade) was added and the mixture stirred at 60 °C for 48 hrs. The reaction was cooled to 20 °C, the solids collected and washed with water (500 ml, HPLC grade), and then the solids were then refluxed in water (1 L, HPLC grade) for 16 hrs. Water was added (200 mL, HPLC grade) and reflux was continued for an additional 16hrs. Water was added (100 mL, HPLC grade) and reflux was continued for an additional 60hrs. The mixture was cooled to 15 °C and solids collected and washed with water (150 mL, HPLC grade). The solids were dried in vacuo to provide title compound as a white solid. Yield. 213.67 g, 662.7 mmol, 82.2%. LCMS m/z 323.3 [M+H]⁺. ¹H NMR (400 MHz, DMSO-c/₆) 6 5.76 (s, 2H), 4.91-5.09 (m, 1 H), 4.69-4.90 (m, 2H), 4.27-4.59 (m, 3H), 3.39-3.56 (m, 2H), 3.29-3.37 (m, 1 H, assumed, partially obscured by H2O), 3.14-3.29 (m, 2H), 2.95-3.13 (m, 1 H), 2.41 (s, 3H), 2.29 (s, 3H), 1.06-1.14 (m, 3H), 0.48 (s, 3H). Example 2 was at least 99.0% chemically pure.

HPLC @210nM 99.62%: The chemical purity was determined using a Waters Acquity UPLC/PDA/TQD instrument. The chromatographic conditions were as follows: Mobile phase A: 10 mM of ammonium bicarbonate in water. Mobile phase B: acetonitrile. Flow rate: 0.5 ml/min. Column: Acquity UPLC BEH C8 1.7 urn, 2.1 mm x 100 mm. Column temperature: 45C. The gradient started at A:B 95%:5% and was linearly increased to 100% B within 8.30 min and held at 100% B for 0.5 min, before it was returned back to the initial gradient of A:B 95%:5% at 8.80 min and was equilibrated at A:B 95%:5% for 1 .5 min. PDA: 200 nm to 400 nm with resolution 1 .2 nm and sampling rate 10 points/sec. The raw data was acquired and processed with Waters Empower 3 software. PDA 210 nm relative peak areas % were obtained with Apex Detection and Integration algorism. The chemical purity % was reported as the relative peak area % of Example 2, with a value of 99.62%. qNMR 99.0 (wt/wt)%: qNMR potency factor (weight percentage) was determined as follows: 6.63 mg of Example 3 was weighed into a pre-tared vial. To this vial, 884.47 mg of an internal standard (Cambridge Isotope Laboratories Inc, Dimethyl Sulfoxide-d6 (D, 99.9%)+0.05% V/V TMS+20 mM Maleic Acid, lot # PR-25603. Potency 0.976) was added. After thorough mixing, the resulting solution was transferred to an NMR tube and the qNMR raw data was acquired using an Oxford AS500 NMR system with the following acquisition parameters: Acquisition Time: 8.1789 sec. Frequency: 500 MHz. Nucleus: ¹H. Pulse Sequence: zg30. SW: 8012.82 Hz. Original Points Count: 65536. The raw data was processed with ACD/Labs 2017 software and the potency factor of Example 2 was calculated using a qNMR potency calculation micro. The potency factor value is 99.0 (wt/wt)%.

CI₆H26N₄O₃: Calculated: C 59.61 , H 8.13, N 17.38. Found (Average for 3 Samples): C 59.47, H 8.13, N 17.29.

Example 3: Preparation of Form 2 of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H- imidazo[4,5-c]pyridin-1-yl)methyl)-2 -methyl propane-1 ,3-diol (PF-07225570)

Step 1 : Synthesis of Intermediate (A): 2,4-Dichloro-5,6-dimethyl-3-nitropyridine

To a room temperature solution of POCI₃ (25.2 ml, 5.0 eq.) was added 5,6-dimethyl-3- nitroporyine-2,4-diol (10.0 g, 54.3 mmol) portion-wise. The resultant mixture was gradually warmed up to 85 °C and held at 85 °C for 3hrs. Once deemed complete by UPLC, the darkened mixture was cooled to room temperature and added slowly to a rapidly stirred slurry of ice/water (250 ml) over 30 minutes. Solids formed upon addition to the ice/water, and the slurry was stirred at room temperature overnight. The solids were filtered and rinsed with water (5 vol, 50 mL). The nearly white solids were dried in an oven at 35 °C under vacuum. The solids were dried to constant weight whereupon Intermediate (A) was isolated as a white solid. Yield. 10.2 g, 13.6 mmol, 85%. ¹H NMR (400 MHz; CDCI₃) 6 2.63 (s, 3H), 2.43 (s, 3H); ¹³C NMR (100 MHz; CDCI₃) 6 159.8, 138.8, 136.5, 130.5, 130.2, 24.3, 14.9.

Step 2: Synthesis of Intermediate (B): 2-Chloro-5,6-dimethyl-3-nitro-A/-((2,2,5-trimethyl-1 ,3- dioxan-5-yl)methyl)pyridine-4-amine.

A solution of Intermediate (A) (40.0 g, 181 mmol) in dimethylacetamide (DMAC) (4.0 vol, 160 ml) was treated with (2,2,5-trimethyl-1 ,3-dioxan-5-yl)methanamine (62.0 g, 362 mmol), triethylamine (54.9 g, 543 mmol) and water (1.0 vol, 40 ml). The slurry was warmed to 35 °C for 48hrs, and once deemed complete by UPLC, was cooled to room temperature. The resultant orange-yellow solids were filtered and washed with water (10 vol, 400 ml). The solids were dried to constant weight whereupon Intermediate (B) was isolated as a yellow-orange solid. Yield. 40.0 g, 116 mmol, 64.3%. ¹H NMR (400 MHz; CDCI₃) 6 5.51 (t, J = 6.0 Hz, 1 H), 3.70 (q, J = 12.0 Hz, 4H), 3.22 (d, J = 4.0 Hz, 2H), 2.47 (s, 3H), 2.18 (s, 3H), 1 .45 (d, J = 4.0 Hz, 6H), 0.85 (s, 3H); ¹³C NMR (100 MHz; CDCI₃) 6 157.3, 146.8, 139.9, 132.0, 118.9, 99.2, 68.1 , 50.6, 33.8, 28.2, 23.2, 19.9, 14.7.

Step 3: Synthesis of Intermediate (C): A/²,A/²-bis(4-methoxybenzyl)-5,6-dimethyl-3-nitro-A/⁴- ((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-pyridin-2,4-diamine

A solution of Intermediate (B) (60.0 g, 175 mmol) in 2-propanol (5 vol, 300ml) was treated with bis(4-methoxybenzyl)amine (135.5 g, 524.0 mmol). The slurry was warmed to 85 °C for 72hrs, and once deemed complete by UPLC, was cooled to room temperature. The resultant yellow solids were filtered and washed with 10 °C, 2-propanol (5 vol, 300 ml). To the solids was added 2-propanol (10 vol, 600 ml), and the resultant solution was warmed to 85 °C where the solids eventually dissolved. The mixture was cooled to room temperature over 4hrs. The yellow solids were filtered and washed with 10 °C, 2-propanol (4.0 vol, 240 ml). The yellow solids were placed in a drying oven at 50 °C until a constant weight was achieved. Intermediate (C) was isolated as a yellow solid. Yield. 87.0 g, 154 mmol, 88.3%. ¹H NMR (400 MHz; CDCI₃) 6 7.08 - 7.05 (m, 4H), 6.84 - 6.81 (m, 4H), 6.47 (t, J = 6 Hz, 1 H), 4.36 (s, 4H), 3.81 (s, 6H), 3.62 (q, J = 12.0 Hz, 4H), 3.44 (d, J = 4 Hz, 2H), 2.37 (s, 3H), 2.23 (s, 3H), 1.45 (s, 3H) 1.42 (s, 3H), 0.85 (s, 3H); ¹³C NMR (100 MHz; CDCh) 6 159.0, 158.6, 153.4, 150.7, 129.6, 125.2, 113.4, 110.3, 98.3, 67.2, 54.9, 51.7, 51.0, 34.1 , 27.6, 24.0, 19.8, 17.9, 15.5.

Step 4: Synthesis of Intermediate (D): 2-(ethoxymethyl)-A/,A/-bis(4-methoxybenzyl)-6,7-dimethyl- 1 -((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-1 /7-imidazo[4,5-c]pyridin-4-amine

Intermediate (D)

To a solution of Intermediate (C) (10.0 g, 17.7 mmol) in tetrahydrofuran (50 ml) at 22 °C was added platinum, 5% on carbon JM Type 5R103 (0.50 g, 0.13 mmol). The reaction vessel was sealed, purged with nitrogen gas three times while stirring, and purged with hydrogen gas three times while not stirring. The reaction vessel was then pressurized to 50 psi, and heated and stirred at 50 °C for 12hrs. The reaction was cooled to 22 °C and purged with nitrogen gas three times. The resulting mixture was filtered to remove platinum on carbon and rinsed with tetrahydrofuran (25 ml). To the filtrate was added 1 M potassium hydroxide (50 ml) and 2-ethoxyacetyl chloride (2.27 ml, 20.4 mmol) at 22 °C. The reaction was stirred at 22 °C for 18hrs. Additional 2- ethoxyacetyl chloride (0.99 ml, 8.9 mmol) was added at 22 °C, and the reaction was stirred for 2hrs. The aqueous layer was removed and extracted with tetrahydrofuran (25 ml). To the combined organics was added 3M potassium hydroxide solution (55 ml). The organic layer was removed by distillation, and isopropanol (55 ml) was added to the reaction mixture. The reaction was then stirred at 90 °C for 40hrs. The reaction was cooled to 22 °C and isopropanol (55 ml) was added. The reaction was heated to 70 °C, until all solids dissolved. At this point, the reaction was cooled to 22 °C at a ramp rate of 0.1 °C/min. The solids were filtered, washed with isopropanol (33 ml) and dried under vacuum to provide Intermediate (D) as a white solid. Yield. 9.70 g, 16.1 mmol, 90.9%.

Step 5: Synthesis of Intermediate (E): 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 H- imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol

To a solution of Intermediate (D) (3.0 g, 4.98 mmol) in toluene (45 ml) at 22 °C was added concentrated HCI (21 ml). The reaction was heated to 75 °C and stirred for 2.5hrs. The reaction was cooled to 22 °C and the organic layer was removed. To the stirring aqueous layer was added 5M aqueous potassium carbonate (25 ml). The reaction was then stirred at 110 °C for 18hrs. The reaction was cooled to 22 °C and water (30 ml) was added. The solids were filtered, washed with water (9 ml) and dried under vacuum to provide the title compound as a white/off-white solid. Yield. 1.49 g, 4.62 mmol, 92.8%.

Step 6: Preparation of pure Form 2 of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7- imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol (PF-07225570)

A solution of Intermediate (E) (1.75 g, 5.43 mmol) in 1 -butanol (10.5 ml) was heated to 60 °C and stirred for 18hrs. At 60 °C, n-heptane (7 ml) was added over 1 hr, and then stirred for 2hrs. The reaction was then cooled to 22 °C over 4hrs. The solids were filtered, washed with a 3:2 1 -butanol: n-heptane mixture (6 ml) and dried under vacuum to provide Form 2 as a white crystalline, anhydrous solid. Yield. 1.47 g, 4.56 mmol, 84.0%.

Powder X-Ray Diffraction:

Powder X-ray diffraction data were collected on Bruker D8 Endeavor diffractometer equipped with a Cu radiation source. The divergence slit was set at 11 mm constant illumination. The sample rotation was set to 15 rpm. Diffracted radiation was detected by a LYNXEYE detector, with the position sensitive detector (PSD) opening set at 3.998 degrees. The X-ray tube voltage and amperage were set to 40 kV and 40 mA respectively. Data was collected in the theta-theta goniometer at the Cu wavelength from 2.0 to 55.0 degrees 2-theta using a step size of 0.019 degrees and a time per step of 0.20 seconds. The sample was prepared by placing the powder in Si low background cavity holder. The sample powder was pressed by a glass slide to ensure that a proper sample height was achieved. Data were collected using Bruker DIFFRAC software and analysis was performed by DIFFRAC EVA software. The PXRD patterns collected were imported into Bruker DIFFRAC EVA software. The peak selection was carried out using EVA software (Version 5.0) function “Peak Search” (Width = 0.278, Threshold = 1) was carefully checked to ensure that all peaks below 30 2-D had been captured and all peak positions had been accurately assigned. The error of ± 0.2° 2-theta in peak positions applies to this data. The minor error associated with this measurement can occur because of a variety of factors including: (a) sample preparation (e.g., sample height), (b) instrument, (c) calibration, (d) operator (including those errors present when determining the peak locations), and (e) the nature of the material (e.g. preferred orientation and transparency errors). Therefore, peaks are considered to have an associated error of ± 0.2° 2-theta. Peaks existing as shoulders, on a higher intensity adjacent peak, have also been removed from the peak list. While the shoulders may be > 0.2° 2-theta from the position of the adjacent peak, they are not considered as discernible from the adjacent peak. To obtain the absolute peak positions, the powder pattern should be aligned against a reference. This was done using the simulated powder pattern from the crystal structure of this solid form solved at room temperature.

A: Powder X-Ray Diffraction for Form 2 as prepared in Example 3:

The PXRD profile of Form 2 is provided in FIG. 1 . Table 1 provides a list of peaks having relative intensity greater than 5% for Form 2.

Table 1 : PXRD peak list for Form 2:

Characteristic PXRD peaks for Form 2 are 8.0, 8.4, and 16.6 °2-Theta ± 0.2 °2-Theta. Characteristic PXRD peaks for Form 2 can also include 21 .6, providing 8.0, 8.4, 16.6, and 21 .6 °2- Theta ± 0.2 °2-Theta. An embodiment of the present invention includes an anhydrous crystalline form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2- methylpropane-1 ,3-diol free base, having a powder X-ray diffraction (PXRD) pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ± 0.2 °2Q. Another embodiment of the present invention includes the anhydrous crystalline form having a PXRD pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ± 0.2 °2Q, and further comprising a peak value of 21 .6 °20 ± 0.2 °20. A single crystal of Form 2 was obtained as presented in FIG. 2. The single crystal was grown by slow evaporation from methanol at room temperature. The single crystal data shows that the crystalline solid is anhydrous.

B: Powder X-Ray Diffraction of PF-07225570 as prepared in Example 2 (Predominantly Form 1 with Form 2 as crystalline impurity): The PXRD profile of PF-07225570 prepared in Example 2 (with Form 1 as predominant crystalline form and with some Form 2 as crystalline impurity) is provided in FIG. 3. Table 2 provides a list of peaks having relative intensity greater than 5% for Form 1 . The impurity peaks of Form 2 are indicated by an asterisk (*) Table 2: PXRD peak list for PF-07225570 as prepared in Example 2, with Form 1 as predominant crystalline form and with Form 2 as an impurity of Form 1. Table shows only peaks with relative intensity > 5%. Form 2 peaks are indicated by an asterisk (*)

Simulated powder pattern of Form 1 was obtained from single crystal structure. Single crystal was grown from acetonitrile. Single crystal data indicates that the material analysed is anhydrous (FIG. 4). A simulated powder pattern from the single crystal structure was obtained via a calculation using Reflex/Powder Diffraction Toolbox in Materials Studio 2018 software package. The simulated pattern from the crystal structure is consistent with the experimentally obtained powder pattern for Form 1 except for several peaks of Form 2 indicated in the Table 2 with an asterisk (*).

Characteristic PXRD peaks for Form 1 comprises 7.5, 9.3, and 15.7 °20 ± 0.2 °20. Characteristic PXRD peaks for Form 1 can also include 9.7, providing 7.5, 9.3, 9.7, and 15.7 °20 ± 0.2 °20. Characteristic PXRD peaks for Form 1 can also include 18.5 °20 ± 0.2 °20, providing

7.5, 9.3, 9.7, 15.7, and 18.5 °20 ± O.2 °20.

The PXRD peak profile for PF-07225570 prepared in Example 2, with Form 1 as predominant crystalline form, clearly shows characteristic peaks of Form 2. For example, the peak 16.6 °20 ± 0.2 °20 has a relative intensity of 26.6%. On the contrary, no characteristic peak of Form 1 , with relative intensity over 5%, shows in the PXRD peak profile for Form 2. Therefore, the present disclosure provides a pure or substantially pure crystalline form of 2-((4-amino-2- (ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base, which can provide better quality control of drug product manufacturing process and drug product compositions.

C: Powder X-Ray Diffraction of Amorphous Form of PF-07225570

The amorphous form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5- c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base was manufactured by cryo-milling. Approximately 430mg Form 2 was loaded into a 5mL steel milling jar with a 7mm steel milling ball, which was then sealed and submerged in liquid nitrogen for 5 minutes. The milling jar was then removed from the liquid nitrogen and placed on a Retsch MM400 ball mill, where the sample was milled for 10 minutes at 30Hz, before being returned to the liquid nitrogen. This process was repeated 4 times, such that the total milling time was 40 minutes.

The PXRD profile collected for the obtained powder provided in FIG. 5 is typical for amorphous material.

Raman Spectroscopy of Amorphous Form of PF-07225570

Raman spectra for amorphous form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7- imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base were collected using a RAM II FT-Raman module attached to a Vertex 70 spectrometer (Bruker Optik GmbH). The instrument is equipped with a 1064 nm solid-state (Nd:YAG) laser and a liquid nitrogen cooled germanium detector. Prior to data acquisition, instrument performance and calibration verifications were conducted using a white light source, and polystyrene and naphthalene references.

Samples were prepared and analysed in truncated NMR tubes. A sample rotator (Ventacon, UK) was used during measurement to maximise the volume of material exposed to the laser during data collection. The backscattered Raman signal from the sample was optimized and data were collected at a spectral resolution of 2 cm^-1 using a laser power of 750 mW. A Blackmann-Harris 4-term apodization function was applied to minimise spectral aberrations. Spectra were generated between 3500 and 50 cm^-1 with the number of scans adjusted accordingly to ensure adequate signal to noise.

Spectra were normalised by to the intensity of the most intense peak to 2.00. Peaks were then identified using the automatic peak picking function in the OPUS v8.2 software (BrukerOptik GmbH) with the sensitivity set to 2%. Peak positions and relative peak intensities were extracted and tabulated. The variability in the peak positions with this experimental configuration is within ±2 cm'¹, unless otherwise stated in Table 3.

It is expected that, since FT-Raman and dispersive Raman are similar techniques, peak positions reported in this document for FT-Raman spectra would be consistent with those which would be observed using a dispersive Raman measurement, assuming appropriate instrument calibration.

Table 3 - Raman peak list of the amorphous form of 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl- 1/7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base. A variability of ±2 cm'¹ is assigned to these peaks, unless otherwise stated.

All publications and patent applications cited in the specification are herein incorporated by reference in their entirety. It will be apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A substantially pure anhydrous crystalline form of 2-((4-amino-2-(ethoxymethyl)-6,7- dimethyl-1 /7-imidazo[4,5-c]pyridin-1-yl)methyl)-2-methylpropane-1 ,3-diol free base, having a powder X-ray diffraction (PXRD) pattern comprising peaks values of 8.0, 8.4, and 16.6 °20 ± 0.2 °20.

2. The substantially pure anhydrous crystalline form of claim 1 , having a PXRD pattern further comprising a peak value of 21 .6 20 ± 0.2 20.

3. The substantially pure anhydrous crystalline form of claim 1 or 2, having at least 95% crystalline purity.

4. The substantially pure anhydrous crystalline form of claim 3, having at least 98% crystalline purity.

5. The substantially pure anhydrous crystalline form of any one of claims 1 to 4, wherein the PXRD pattern is free of peaks with a relative intensity over 5% selected from the group consisting of 7.5, 9.3, and 15.7 °20 ± 0.2 °20.

6. A pharmaceutical composition comprising the substantially pure anhydrous crystalline form of any one of claims 1 to 5, and a pharmaceutically acceptable carrier or excipient.

7. A method for the treatment of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the substantially pure anhydrous crystalline form of any one of claims 1 to 5.

8. The method of claim 7, wherein the substantially pure anhydrous crystalline form is administered with at least one additional therapeutic agent.

9. The substantially pure anhydrous crystalline form of any one of claims 1 to 5, for use in the treatment of cancer in a subject in need thereof.

10. A method of preparing the substantially pure anhydrous crystalline form of any one of claims 1 to 5, wherein the method comprises treating 2-(ethoxymethyl)-N,N-bis(4- methoxybenzyl)-6,7-dimethyl-1-((2,2,5-trimethyl-1 ,3-dioxan-5-yl)methyl)-1 H-imidazo[4,5- c]pyridin-4-amine in a solvent system comprising a solvent with a boiling point of at least 70 °C, with an acid to provide 2-((4-amino-2-(ethoxymethyl)-6,7-dimethyl-1 /7-imidazo[4,5-c]pyridin-1-

29 yl)methyl)-2-methylpropane-1 ,3-diol as a solid, wherein the anhydrous crystalline form of any one of claims 1 to 5 is predominant crystalline form in the solid.

11. The method of claim 10, wherein the method further comprises dissolving the solid in a solvent system comprising a C1-10 alcohol to form a solution and heating the solution at an elevated temperature; and followed by adding a solvent comprising a C4-10 alkane to the solution to provide the substantially pure anhydrous crystalline of any one of claims 1 to 5.

12. The method of claim 11 , wherein said C1-10 alcohol is 1 -butanol.

13. The method of claim 11 or 12, wherein said C4-10 alkane is n-heptane.

14. The method of any one of claims 10 to 13, wherein said solvent with a boiling point of at least 70 °C is toluene.

15. The substantially pure anhydrous crystalline form of any one of claims 1 to 5, wherein the substantially pure anhydrous crystalline form is prepared by the method of any of claims 10 to 14.

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